Holographic Sensor

ABSTRACT

A sensor for the detection of an analyte comprising a cis-diol moiety, which comprises a holographic element comprising a medium and a hologram disposed throughout the volume of the medium, wherein an optical characteristic of the element changes as a result of a variation of a physical property occurring throughout the volume of the medium, and wherein the medium is a polymer comprising a group of formula (i) 
     
       
         
         
             
             
         
       
     
     wherein
         n is 0, 1, 2, 3 or 4;   each X (if present) is independently is an atom or group which, via an electronic effect, promotes formation of a tetrahedral geometry about the boron atom; and   Y is a spacer which, when n is 0 or otherwise optionally, is an atom or group which, via an electronic effect, promotes formation of a tetrahedral geometry about the boron atom. Such a sensor may be used for the detection of glucose.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation application of co-pending applicationSer. No. 10/545,275, filed Oct. 17, 2005, now U.S. Pat. No. 8,048,680,which was filed as a National Stage Application of InternationalApplication Number PCT/GB2004/000576, filed Feb. 13, 2004; which claimspriority to Great Britain Application No. 0305587.8, filed Mar. 11,2003; all of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to a holographic sensor.

BACKGROUND OF THE INVENTION

WO-A-95/26499 discloses a holographic sensor, based on a volumehologram. The sensor comprises a holographic element, the elementcomprising an analyte-sensitive holographic support medium having anoptical transducing structure disposed throughout its volume. Because ofthis physical arrangement of the transducer, the optical signalgenerated by the sensor is very sensitive to volume changes orstructural rearrangements taking place in the analyte-sensitive matrixas a result of interaction or reaction with the analyte. For example, asensor comprising a gelatin-based holographic medium may be used todetect trypsin. Trypsin acts on the gelatin medium, irreversiblydestroying the integrity of the holographic support medium.

WO-A-03/087899 describes a method for the continuous detection of ananalyte in a fluid involving the use of a holographic sensor. Thesupport medium of the sensor comprises a group which is capable ofreacting reversibly with the analyte. Thus, when fluid is passed overthe holographic element, any analyte present can be detectedcontinuously.

In particular, WO-A-03/087899 describes how a holographic sensor formedby the polymerisation of monomers including vinylphenylboronic acid maybe used to detect glucose. The pendant phenylboronic acid groups canreact reversibly with a cis-diol group of glucose, resulting in swellingof the holographic support medium. A limitation of this technology isthat the sensor is only effective over a limited pH range; there remainsthe need for sensors which can detect glucose continuously over a rangeof conditions, in particular physiological conditions.

SUMMARY OF THE INVENTION

When a cis-diol-containing species binds a boronic acid, RB(OH)₂, anunstable boronate ester results, the ester having a trigonal planarconformation. The boronate ester normally achieves stability by bindingan electron-donating group, to form a more stable, tetrahedral geometry.Typically, boronic acids attain this tetrahedral geometry by bindingOH⁻, forming negatively-charged boronate esters. At relatively high pH,the mechanism is believed to be slightly different. It is thought thatthe boronic acid first binds OH⁻, to form tetrahedral RB(OH)₃ ⁻, whichthen reacts with the cis-diol. The tetrahedral RB(OH)₃ ⁻ reacts morereadily with a cis-diol than the trigonal planar boronate ester.

Without wishing to be bound by theory, the inventors believe that theglucose sensor of WO-A-03/087899 “works” because the formation of anegatively-charged phenylboronate ester produces a Donan potential,causing water to partition into the support medium. Expansion of themedium is then observed as a shift in the reflectance maxima to longerwavelengths. At low pH values, the boronic acid groups may be unable toform negatively-charged phenylboronate esters and, as a result,detection might not be possible. This is probably why the glucose sensorof WO-A-03/087899 is only effective over a limited pH range.

The invention is based on the discovery of a class of phenylboronic acidderivatives which allow for the detection of glucose and othercis-diol-containing analytes across a wide range of pH values. Theinventors have realised that phenylboronic acids can be modified topromote formation of a more reactive tetrahedral conformation.

For example, the phenyl group may comprise one or electron-withdrawingsubstituents which, by mediating their electronic effects through thephenyl ring, promote formation of RB(OH)₃ ⁻. As another example, asubstituent may be capable of forming an intramolecular bond with theboron atom, forcing the boronate into a substantially tetrahedralconformation. Judicial selection of substituents allows theresponsiveness of the sensor to be optimised with respect to aparticular set of detection conditions.

Accordingly, a first aspect of the invention is a sensor for thedetection of an analyte comprising a cis-diol moiety, which comprises aholographic element comprising a medium and a hologram disposedthroughout the volume of the medium, wherein an optical characteristicof the element changes as a result of a variation of a physical propertyoccurring throughout the volume of the medium, and wherein the mediumcomprises a polymer comprising a group of formula (i)

wherein

n is 0, 1, 2, 3 or 4;

each X (if present) is independently an atom or group which, via anelectronic effect, promotes formation of a tetrahedral geometry aboutthe boron atom; and

Y is a spacer which, when n is 0 or otherwise optionally, is an atom orgroup which, via an electronic effect, promotes formation of atetrahedral geometry about the boron atom.

Such a sensor can be used in a method for the detection of an analytecomprising a cis-diol moiety in a fluid, which comprises contacting thefluid with the holographic element and detecting any change of theoptical characteristic of the element. The analyte may comprise aplurality of cis-diol moieties; examples of such analytes includeglucose and tartaric acid (tartarate).

The polymeric medium may be obtained by the polymerisation of monomersincluding a compound of formula (I)

wherein

X, Y and n are as defined above; and

Z is a polymerisable group.

Another aspect of the invention is a device for the detection of ananalyte comprising a cis-diol moiety in a fluid, which comprises a fluidconduit having an inlet, an outlet, and a holographic element as definedabove over which the fluid can flow, wherein the device also includes awindow whereby non-ionising radiation can irradiate the holographicelement. The analyte concentration may change, while the fluid isstatic. Alternatively, the fluid may be passed continuously over theelement.

The variation arises as a result of reaction between the medium and thecis-diol moiety of the analyte, wherein the reaction and the variationare reversible. Since both the reaction and the reverse reaction canoccur, analytes such as glucose can be continuously detected, possiblyin real time

A sensor of the invention may be used to monitor a reaction in vivo orin vitro, e.g. in a fermenter. It can be used for kinetic measurement,and as an effective control system. The sensor may be used, for example,to detect hyperglycaemia or hypoglycaemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the response of the hologram in terms of peak diffractionwavelength readings.

FIG. 2 shows a calibration curve.

FIG. 3 shows that the addition of salt had no significant effect on thehologram, showing that it could easily tolerate small changes in saltconcentrations.

FIG. 4 shows a calibration curve for response to glucose in PBS pH 7.4at 30° G.

FIG. 5 shows a calibration curve.

FIG. 6 shows the response of the sensor to the five analytes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In addition to their use in holographic sensors, the present inventionalso encompasses the novel phenylboronic acid compounds themselves.Certain compounds and combinations of substituents are preferred; inparticular, see the subclaims.

The term “alkyl” as used herein refers to a straight or branched chainalkyl moiety having from one to six carbon atoms. The term includes, forexample, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl,hexyl and the like.

The term “alkoxy” as used herein refers to a straight or branched chainalkoxy moiety having from one to six carbon atoms. The term includes,for example, methoxy, ethoxy, propoxy and the like.

The term “halogen” as used herein refers to fluorine, chlorine, bromineor iodine.

The term “electronic effect” as used herein refers to a direct orindirect effect on the boronic acid group, which promotes formation of atetrahedral conformation about the boron atom relative to phenylboronicacid. The atom or group may, for example, have an electron-withdrawing,electron-donating, resonance or mesomeric effect on the phenyl ring offormulae (i) and (I) which, in turn, effects the boronic acid group.

The phenyl ring is preferably substituted with one or moreelectron-withdrawing groups. In this way, formation of RB(OH)₃ ⁻ may bepromoted. When RB(OH)₃ ⁻ reacts with a cis-diol group of glucose, theresulting negatively-charged phenylboronate ester produces a Donanpotential, causing water to partition into the support medium. Expansionof the medium is then observed as a shift in the reflectance maxima tolonger wavelengths. In general, most sensors of the invention willdetect cis-diol-based analytes in this way.

Particularly when the analyte comprises a plurality of cis-diol groups,it is preferred that the phenyl ring is substituted with a groupcomprising an atom having a lone pair of electrons, which can form anintramolecular (e.g. coordinate) bond with the boron atom, forcing itinto a tetrahedral conformation. The boronic acid group may, as aresult, be highly reactive. An example of such a group is illustratedbelow:

Although such a group is highly reactive to cis-diols, it is thought toform an uncharged phenylboronate ester which, as it is uncharged, cannotproduce a Donan potential. Instead, it is believed that, when theanalyte comprises a plurality of cis-diol moieties, it can bind two ofthese highly reactive phenylboronic acid groups and effectivelycross-link the support medium. This cross-linking of the support mediumcauses it to contract, resulting in a shift in the replay wavelength.Examples of analytes which can be detected in this way are glucose andtartarate (tartaric acid).

The interaction between the medium and analyte can be detected remotely,using non-ionising radiation. The extent of interaction is reflected inthe degree of change of the physical property, which is detected as avariation in an optical characteristic, preferably a shift in wavelengthof non-ionising radiation.

The property of the holographic element which varies may be its chargedensity, volume, shape, density, viscosity, strength, hardness, charge,hydrophobicity, swellability, integrity, cross-link density or any otherphysical property. Variation of the or each physical property, in turn,causes a variation of an optical characteristic, such as polarisability,reflectance, refractance or absorbance of the holographic element.

The hologram may be disposed on or in, part of or throughout the bulk ofthe volume of the support medium. An illuminating source of non-ionisingradiation, for example visible light, may be used to observevariation(s) in the, or each, optical characteristic of the holographicelement.

More than one hologram may be supported on, or in, a holographicelement. Means may be provided to detect the or each variation inradiation emanating from is the or each hologram, arising as a result ofa variation in the or each optical characteristic. The holographicelements may be dimensioned and arranged so as to sense two or moreindependent events/species and to affect, simultaneously, or otherwise,radiation in two or more different ways. Holographic elements may beprovided in the form of an array.

Preferred groups of formula (i) include:

The holographic support medium may be obtained by the polymerisation ofmonomers, wherein the monomers include a compound of formula (I)Preferred monomers include:

-   2-(4-(acrylamidomethyl)phenylamino)methyl)phenylboronic acid;-   2-((3-methacrylamidopropylamino)methyl)phenylboronic acid;-   2-acrylamido-phenylboronic acid;-   3-acrylamido-phenylboronic acid; and-   3-acrylamido-6-fluoro-phenylboronic acid.

In addition to a compound of formula (I), the monomers may include(meth)acrylamide and/or (meth)acrylate-derived co-monomers. Inparticular, the monomer HEMA (hydroxyethyl methacrylate) is readilypolymerisable and cross-linkable. PolyHEMA is a versatile supportmaterial since it is swellable, hydrophilic and widely biocompatible.The monomers may also include co-monomers having groups which arecapable of intermolecular electron-donation, for example secondary oftertiary amines.

Other examples of holographic support media which may be modified toinclude boronate group are gelatin, K-carageenan, agar, agarose,polyvinyl alcohol (PVA), sol-gels (as broadly classified), hydro-gels(as broadly classified), and acrylates.

A parameter determining the response of a holographic element is theextent of cross-linking. The number of cross-linking points due topolymerisation of monomers should not be so great that complex formationbetween polymer and analyte-binding groups is relatively low, since thepolymer film may become too rigid. This may inhibit the swelling of thesupport medium.

The following Examples illustrate the invention, in conjunction with theaccompanying drawings.

Example 1

3-Acrylamido-phenylboronic acid (“3-APB”) was synthesised by reacting3-amino phenylboronic acid with an excess of acryloyl chloride in anaqueous alkaline solution. The product was extracted in acetone anddried using a rotary evaporator. The structure of 3-APB was confirmedusing NMR. The purity was about 90%, TLC showing very littlecontamination.

3-APB was then copolymerised with acrylamide and N,N′-methylenebisacrylamide, and a hologram recorded within the polymer material. Theresponse to glucose was then tested by increasing the glucoseconcentration in phosphate-buffered saline (PBS) solution at pH 7.4 in0.5 mM steps.

FIG. 1 shows the response of the hologram in terms of peak diffractionwavelength readings. The reaction with glucose was fully reversibleafter the system was flushed twice with fresh buffer. The sensor wasalso sensitive enough to pick up concentrations of glucose as low as 0.5mM (9 mg %) with a shift of about 6 nm. This result was highlyreproducible with errors of about 5%, even when using a differenthologram and instrumentation.

FIG. 2 shows the calibration curve. The calibration was approximatelylinear below 2 mM (36 mg %) glucose.

A control was also run where 2 mM KCl was added to the system containingthe hologram in PBS pH 7.4 instead of glucose. This was to test thetolerance of the hologram to changes in osmolarity; an increase inosmolarity could lead to a contraction of the polymer. As shown in FIG.3, addition of the salt had no significant effect on the hologram,showing that it could easily tolerate small changes in saltconcentration.

Example 2

3-APB, the synthesis of which is described in Example 1, wasrecrystallised from an aqueous ethanolic solution with a purity of about98%; both NMR and TLC showed that there were no contaminants present.

3-APB was co-polymerised with acrylamide and N,N′-methylenebisacrylamide to form a polymer comprising about 15 mole % of purified3-APB and about 1.55 mole % N,N′-methylene bisacrylamide (cross-linker).A hologram was then recorded in the polymer.

A calibration curve for response to glucose in PBS pH 7.4 at 30 C. isshown in FIG. 4. The purified 3-APB had a response of about 14 nm permillimolar glucose whereas the 90% pure 3-APB of Example 1 had aresponse of only 11 nm per millimolar glucose for a hologram with thesame mole % of 3-APB.

A polymer comprising a 3-APB molar fraction of 25% was synthesised usingthe same amount of cross-linker (1.55 mole %) and the same amount ofsolids per unit volume of solvent. A hologram was recorded within thepolymer and then calibrated. The calibration curve is shown in FIG. 5.

The increased amount of 3-APB increased the sensitivity of the hologramfor glucose by over 70%. This allowed small changes in glucoseconcentration to be accurately detected.

Example 3

2-Acrylamido-phenylboronic acid (“2-APB”) was synthesised by reacting2-aminophenylboronic acid with an excess of acryloyl chloride in anaqueous alkaline solution. The product was extracted in acetone anddried using a rotary evaporator. The structure of 2-APB was confirmedusing NMR. The purity was shown to be greater than 90%.

2-APB was then copolymerised with acrylamide and N,N′-methylenebisacrylamide to form a co-polymer with 20% 2-APB and 1.5%N,N′-methylene bisacrylamide (cross-linker). A hologram was thenrecorded within the polymeric medium. The resulting holographic sensorwas then tested for its response to glycerol, ethylene glycol, lactate,tartaric acid and glucose. Testing was conducted using PBS (pH 7.4) at30° C.

The response of the sensor to the five analytes is shown in FIG. 6. Itis evident that the sensor is unresponsive to changes in glycerol,ethylene glycol and lactate concentration. The sensor is, however,sensitive to change in the levels of tartaric acid and glucose; this wasobserved as a blue shift in the peak diffraction wavelength, indicatingthat the support medium contracted in the presence of these analytes.

This selectivity to tartaric acid and glucose is believed to beattributable to the fact that both these analytes contain two cis-diolgroups; the other analytes tested contain only one. Thus, tartaric acidand glucose can bind two 2-APB groups and, effectively, cross-link theholographic support medium, causing it to contract.

The response to tartaric acid is greater than for glucose because thetwo cis-diol sites of tartarate are identical and thus of equal affinityfor 2-APB. The cis-diol sites of glucose are slightly different. If sucha sensor were to be used to monitor physiological levels of glucose,then the greater response to tartaric acid would not be a problem sincethe latter is not found free in solution in the body.

1. A sensor for the detection of an analyte comprising a cis-diolmoiety, which comprises a holographic element comprising a medium and ahologram disposed throughout the volume of the medium, wherein anoptical characteristic of the element changes as a result of a variationof a physical property occurring throughout the volume of the medium,and wherein the medium comprises a polymer comprising a group of formula(i)

wherein X is an atom or group which, via an electronic effect, promotesformation of a tetrahedral geometry about the boron atom of the —B(OH)₂group; and Y is a linker to the remainder of the polymer.
 2. The sensoraccording to claim 1, wherein X is electron-withdrawing.
 3. The sensoraccording to claim 2, wherein X is selected from the group consisting ofhalogen, nitro and cyano.
 4. The sensor according to claim 3, wherein Xis fluorine.
 5. The sensor according to claim 1, wherein X comprises—C(O)— and/or —NH—.
 6. The sensor according to claim 1, wherein X is inthe ortho position relative to the —B(OH)₂ group.
 7. The sensoraccording to claim 6, wherein X comprises an amide or amine group whichis capable of forming an intramolecular bond to the —B(OH)₂ group,thereby forming a ring.
 8. The sensor according to claim 6, wherein X iscapable of forming an intramolecular bond to the —B(OH)₂ group via aresonance or mesomeric effect.
 9. The sensor according to claim 6,wherein X is capable of forming an intramolecular bond to the —B(OH)₂group via lone pair donation.
 10. The sensor according to claim 9,wherein the lone pair donation is from a nitrogen atom.
 11. The sensoraccording to claim 9, wherein the lone pair donation is from an oxygenatom.
 12. The sensor according to claim 11, wherein the oxygen atom is acarbonyl oxygen.
 13. A sensor for the detection of an analyte comprisinga cis-diol moiety, which comprises a holographic element comprising amedium and a hologram disposed throughout the volume of the medium,wherein an optical characteristic of the element changes as a result ofa variation of a physical property occurring throughout the volume ofthe medium, and wherein the medium comprises a polymer comprising agroup of formula (ii)

wherein Y is a spacer providing a link to the remainder of the polymer,and which comprises an amide or amine group which is capable of formingan intramolecular bond to the —B(OH)₂ group, thereby forming a ring. 14.The sensor according to claim 13, wherein said intramolecular bond isformed via a resonance or mesomeric effect.
 15. The sensor according toclaim 13, wherein said intramolecular bond is formed via lone pairdonation.
 16. The sensor according to claim 15, wherein the lone pairdonation is from a nitrogen atom.
 17. The sensor according to claim 15,wherein the lone pair donation is from an oxygen atom.
 18. The sensoraccording to claim 17, wherein the oxygen atom is a carbonyl oxygen.